Project Summary The goal of this project is to provide the candidate with the necessary skills and training to become an independent clinician-scientist in the field of ocular surface mucin glycobiology. The applicant will learn methodologies to analyze high molecular weight O-glycosylated ocular mucins. Analysis of these mucins is challenging due to the extensive branching and numerous glycan attachments and is compounded by the limited sample size obtainable in vivo. Therefore, training of the applicant in this field would be highly valuable and beneficial to the ocular surface community. Mucins on the ocular surface are found both in the tear film and attached to the cornea and bulbar conjunctiva to primarily lubricate the eye. Membrane associated mucins (MAMs) in the conjunctiva form the glycocalyx which is important for hydrophilic attraction of the tears. In dry eye disease (DED), there are changes in the expression of mucins in the cornea and bulbar conjunctiva that contribute to its pathogenesis. While dryness on the ocular surface is thought to lead to increased friction during blinking, the impact of dryness on the epithelial cell physiology of the inner surfaces of the eyelid (palpebral conjunctiva) is unclear. Experiments in Aim 1 will determine whether MAMs MUC1 and MUC16 are present in the palpebral conjunctiva. Experiments in Aim 2 will compare gene expression and protein levels of MUC1 and MUC16 in the palpebral conjunctivas of humans with moderate to severe DED. Finally, experiments in Aim 3 will investigate the affinity of MUC16 for galectin-3 in the glycocalyx. Aims 1 and 2 will use real time quantitative PCR analysis to detect expression of MUC1 and MUC16. The cell samples will be collected using a technique known as impression cytology and then processed for gene expression and protein quantification. Aim 3 will utilize an adapted slot blot affinity assay to analyze the affinity of MUC16 for galectin-3, a lectin that associates with MUC16 in the glycocalyx and contributes to its barrier function. To accomplish these aims, the applicant has assembled a strong mentoring team. The primary mentor, Dr. Jason Nichols, has an extensive history of clinical research, mentoring experiences, and analytical expertise. The co-mentor, Dr. Pablo Argeso, has been researching ocular surface mucins for over a decade and knows the intricacies involved with analysis of these glycoproteins. This K23 supported research will form the foundation for future research into the glycobiological basis of dry eye disease and the impact of ocular inflammation on mucins.